In Vivo Comparison of Site-Specific and Site-Selective Methods for Pretargeted Imaging in a Murine Model of Colorectal Cancer

Pretargeting based on the inverse electron-demand Diels-Alder reaction between a tetrazine and a <i>trans</i>-cyclooctene-modified (TCO) antibody has emerged as a powerful approach to the imaging and therapy of cancer. However, poorly constructed immunoconjugates with TCOs in ill-defined places often leads to suboptimal image contrast in pretargeted immune-PET and SPECT. To address these limitations, site-specific and site-selective approaches to antibody bioconjugation may be used for pretargeted imaging. Herein, we present a comparative study between stochastically, site-specifically, and site-selectively modified immunoconjugates for pretargeted SPECT. First, a trio of immunoconjugates─TCO-CC49, TCO-^Gly(Az)CC49, TCO-^PFP(Az)CC49─were produced with active TCO moieties sufficient for tetrazine ligation. All immunoconjugates retained high affinity for their target antigen, TAG-72, in enzyme-linked immunosorbent assay experiments and in antigen-expressing tumor tissue. A DOTA-modified tetrazine was labeled with indium-111 in high radiochemical yield and used for in vivo experiments. Then, mice bearing subcutaneous LS 174T xenografts received either TCO-CC49, TCO-^Gly(Az)CC49, TCO-^PFP(Az)CC49 followed 72 h later by [¹¹¹In]In-DOTA-Tz (16-20 MBq). SPECT scans were acquired at 1 and 24 h postinjection of radiotracer, and all three constructs produced clear tumor delineation. Ex vivo biodistribution data at 24 h showed [¹¹¹In]In-DOTA-Tz-TCO-CC49 with the highest tumoral uptake (10.4 ± 1.2% ID/g) probably due to the highest TCO loading, but [¹¹¹In]In-DOTA-Tz-TCO-^Gly(Az)CC49 and [¹¹¹In]In-DOTA-Tz-TCO-^PFP(Az)CC49 increased tumor-to-blood and tumor-to-lung ratios. Ultimately, this comparative study serves as a critical step toward identifying the optimal bioconjugation strategy that may be used in pretargeted systems.